| Abstract
| - The conformation of microtubule-bound paclitaxel has been examined by fluorescence andsolid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3‘-N-debenzoyl-3‘-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promotingactivity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino groupdid not adversely affect the ligand−receptor association. The distance between the fluorophore N-AB-PTand the colchicine binding site on tubulin polymers was determined through time-resolved measurementsof fluorescence resonance energy transfer to be 29 ± 2 Å. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the Stokes shift as a functionof solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in proticsolvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokesshift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from theeffects of the solvent. These data are best explained by considering the solution-state conformationalproperties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the natureof the solvent, and these fluorescence data are consistent with the molecule adopting a “hydrophobiccollapsed” conformation in protic solvents and an “extended” conformation in aprotic solvents. The Stokesshift of microtubule-bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was alsoinvestigated by solid-state NMR. Paclitaxel was labeled with 19F at the para position of the C-2 benzoylsubstituent and with 13C and 15N in the side chain. Distances between the fluorine and carbon nuclei weredetermined by REDOR. The distance between the fluorine and the 3‘-amide carbonyl carbon was 9.8 ±0.5 Å, and the distance between the fluorine atom and the 3‘-methine carbon was 10.3 ± 0.5 Å. Thesespectroscopic data were used in conjunction with molecular modeling to refine the microtubule-boundconformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxelbinding site.
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